Monoclonal antibodies to the human ldl receptor, their production and use

ABSTRACT

There are provided monoclonal antibodies to the LDL receptor which are useful for the identification and purification of LDL and in treatment of e.g. hepatitis C infection.

FIELD OF THE INVENTION

[0001] The present invention relates to monoclonal antibodies whichspecifically recognise the human receptor for low-density lipoproteins(LDLR). These antibodies are useful e.g. for the identification andpurification of human soluble LDLR (hsLDLR) in production processes aswell as in the identification and treatment of diseases such as,hepatitis C infection (HCV).

BACKGROUND OF THE INVENTION

[0002] Cholesterol, a component of all eukaryotic plasma membranes, isessential for the growth and viability of cells in higher organisms.However, high serum levels of cholesterol cause disease and death bycontributing to the formation of atherosclerotic plaques in arteriesthroughout the body. The major site of cholesterol synthesis in mammalsis the liver. Appreciable amounts of cholesterol are also formed by theintestine. The rate of cholesterol formation by these organs is highlyresponsive to the amount of cholesterol absorbed from dietary sources.Cells outside of the liver and intestine acquire cholesterol from theplasma rather than by synthesising it de novo. Cholesterol and otherlipids are transported in body fluids by lipoproteins, which areclassified according to increasing density. A lipoprotein is a particleconsisting of a core of hydrophobic lipids surrounded by a shell ofpolar lipids and apoproteins. These lipoproteins have two roles: theysolubilize highly hydrophobic lipids and they contain signals thatregulate the movement of particular lipids in and out of specific targetcells and tissues. Cholesterol is transported in body fluids bylow-density lipoproteins (LDL) which binds to a specific receptor on theplasma membrane of non hepatic cells. The receptor-LDL complex is theninternalised into the cells by a transport mechanism known as receptormediated endocytosis (Goldstein et al. 1979). The low densitylipoprotein (LDL) receptor is the prototype of a family of structurallyrelated cell surface receptors that mediate endocytosis of multipleligands in mammalian cells.

[0003] The LDL receptor consists of 822 amino acid residues and exhibitsa molecular weight of 164000. It is composed of several domains some ofwhich share sequence homology with other proteins. Its NH₂-terminalligand-binding domain consists of 292 residues, arranged in 7cysteine-rich imperfect repeats. Each repeat contains six cysteineresidues which are disulphide bonded in the pattern one to three, two tofive, and four to six. (Bieri et al. 1995). This domain is followed byfour additional domains: the first consists of 400 amino acid residuesand is homologous to the EGF receptor, the second consists of 58 aminoacid residues rich in O-linked sugars, the third is a singletrans-membrane domain of 22 amino acid residues and the fourth is acytoplasmic domain of 50 amino acid residues (Sudhofet al. 1985), (Brownet al. 1986).

[0004] The physiologic importance of the LDL receptor was revealed byBrown and Goldstein's studies on familial hypercholesterolemia (FH). Thedisease was found to be due to a molecular genetic defect resulting inthe absence or deficiency of functional receptors for LDL (Brown et al.1976). Several classes of FH mutations have been characterised.(Goldstein et al. 1975).

[0005] A soluble form of the sLDLR exhibiting antiviral activity wasidentified and isolated from the culture supernatant ofinterferon-induced cells (Fischer et al. 1993) and in body fluids(Fischer et al. 1994). Several interferon-induced proteins have beenidentified that are instrumental in the induction of the antiviral stateby IFNs. One such protein exhibiting antiviral activity was produced andaccumulated in the culture supernatant of human amnion WISH cells. Thisprotein was purified to homogeneity and identified as the sLDLR (see EP0 553 667 and Fischer et al. 1993). The sLDLR was found to be secretedinto the medium by mammalian cells that enter an antiviral state inresponse to interferon. In contrast to interferon, sLDLR does not inducean antiviral state in the cells but is antiviral by itself. It was foundthat sLDLR apparently has to be present throughout the process of viralreplication maturation and budding suggesting it might be involved in acomplex process that leads to the inhibition of virus assembly orbudding (unpublished data). Endocytosis of the hepatitis C virus hasbeen recently shown to be mediated by LDL receptors on cultured cells(Agnello et al. 1999). These and other findings suggest that the familyof LDL receptors may serve as viral receptors. Therefore, antibodiesrised against the sLDLR receptor may block the entry and budding ofviral particles by binding to the cellular LDL receptor. The onlyavailable monoclonal antibody to LDLR known so far is C7, an antibody tobovine LDLR (Beisiegel et al. 1981, commercially available fromAmersham, UK) which was prepared by immunization of mice with the bovineadrenal cortex LDLR purified to homogeneity. Membranes from the bovineadrenal cortex were solubilized and the receptor was partially purifiedby elution from a DEAE-cellulose column (Beisiegel et al. 1981). Theantibody to the bovine LDLR only weakly cross-reacts with human LDLR.

[0006] In fact, the C7 Mab to bovine LDLR was found to have significantdisadvantages when used for detection and quantitation of recombinanthuman LDLR:

[0007] a) It has very low affinity to the human LDLR

[0008] b) It significantly cross reacts with cell culture derivedimpurities

[0009] Specific antibodies to human LDLR were not previously available.This appears surprising since it is very common to raise antibodiesagainst novel proteins, be it for purification, identification or forassay development purposes. It is possible that such antibodies have notbeen generated so far, since a condition for generating monoclonalantibodies is the availability of sufficiently large amounts of highlypurified antigen which allow efficient immunization of mice. A highlypurified antigen is one which appears as a single major peak in RP-HPLC.Furthermore methods for identification and quantitation of the antigenduring purification processes were not easy to establish. In accordancewith the invention, the antiviral activity assay described herein wasemployed for the identification of LDLR during purification processes.

[0010] There exists a need to generate specific Mabs to human solubleLDLR to provide the means for developing an efficient immunoassay(ELISA) and for the identification of the protein in Western blot. Theseantibodies are required for the monitoring and quantitation of therecombinant human soluble LDLR during development of the production andpurification processes of the recombinant protein and for detection ofthe natural protein.

SUMMARY OF THE INVENTION

[0011] The present invention allows the generation of hybridoma cellslines producing monoclonal antibodies capable of specificallyrecognising and binding the human LDL receptor and fragments thereof.

[0012] More specifically the present invention allows the generation ofhybridoma cells lines producing monoclonal antibodies capable ofspecifically recognising and binding the human soluble LDL receptor.

[0013] Thus the present invention relates to a monoclonal antibody,chimeric antibody, humanized antibody, anti-anti-Id antibody or fragmentthereof which specifically recognises and binds the human LDL receptorand fragments thereof, except monoclonal antibody C7.

[0014] The present invention provides such monoclonal antibodies thatrecognise and bind the human soluble LDLR and meet the following needs:

[0015] 1. Mabs that can be used as a pair in an ELISA, e.g. a sandwichELISA (Enzyme Linked Immuno Sorbent Assay) for detection of humansoluble LDLR.

[0016] 2. Mabs that can be used for identification of the LDLR inWestern Blot analysis.

[0017] 3. Mabs that can be used to neutralise the antiviral biologicalactivity of the human soluble LDLR.

[0018] 4. Mabs that can be used to inhibit virus infection, such as HCV.

[0019] The present invention further provides a method for the detectionand/or the quantitation of human LDLR which comprises the use of thespecific monoclonal antibodies according to the invention in a knownmanner for that purpose.

[0020] The present invention also provides cloned hybridoma comprising aspleen cell from a mammal immunized with recombinant human LDLR and ahomogenic or heterogenic lymphoid cell.

[0021] A monoclonal antibody according to the invention is prepared in aconventional manner, e.g. by growing a cloned hybridoma comprising aspleen cell from a mammal immunized with hsLDL and a homogenic orheterogenic lymphoid cell in liquid medium or mammalian abdomen to allowthe hybridoma to produce and accumulate the monoclonal antibody.

[0022] The invention, in yet another aspect, provides a method forpurifying the human LDLR which comprises contacting a materialcontaining crude LDLR with a monoclonal antibody according to theinvention. A column with adsorbed LDLR specific monoclonal antibody maybe used as an affinity purification step, in the purification process ofthe recombinant protein.

[0023] A method for detecting and measuring recombinant human LDLR whichcomprises using as antibody the monoclonal antibodies of the presentinvention in an ELISA assay as described in example 5.

[0024] As the LDLR or fragment of a LDLR for immunizing animals any LDLRcan be used as long as it is the LDLR of a warm-blooded mammal. A muteinof LDLR can be also used. A representative example of such a mammalianhuman soluble LDLR is the soluble LDLR +291 form which includes theamino acid sequence beginning at amino acid Asp at position +4 andending with amino acid Glu at position +291 of the sequence of the humanLDLR, any other form may be used as well, such as the +292 form etc.

BRIEF DESCRIPTION OF THE FIGURES

[0025]FIG. 1 shows a flow chart depicting the development of monoclonalantibodies to r-hsLDLR.

[0026]FIG. 2 shows a Western blot analysis of the +291 form of r-hsLDLRin lane 1, the urinary hsLDLR in lane 2 and recombinant human p55 TNFreceptor as a negative control (r-hTBP-1) in lane 3, with the monoclonalantibodies indicated beneath each strip. The arrows to the left of thefigure indicate the position of the molecular weight markers and thearrows to the right of the figure point to the position of the hsLDLRform indicated above each arrow.

[0027]FIG. 3 shows the effects of Mabs 12.6, 28 and 29.8 on theproduction of HCV(+) and (−) strands in culture FT167. Cells weretreated 30 minutes before infection with MAb anti-LDLR (8 or 2 μg/ml).Then, cells were infected overnight with 25 μl of HCV(+) serum (N°42;1b). The day after Infection, three washes were performed and newmedium was added and changed every 48 hours. Five days after infection,the hepatocytes were harvested, RNA was purified and 1 μg cellular RNAwas analyzed by rTth RT-PCR (Perkin Elmer). Assays were performed induplicate.

[0028] +SP: positive-strand RNA assay; −SP: negative-strand RNA assay;X:blank.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Monoclonal antibodies (Mabs) to human soluble LDLR (hsLDLR) weregenerated. Using these monoclonal antibodies, an ELISA and a Westernblotting procedure for the identification of hsLDLR and a neutralisingassay to the antiviral activity of hsLDLR were developed.

[0030] The Mabs were generated in mice, immunized with the recombinant+291 form of hsLDLR, which consists of the N-terminal ligand bindingdomain of the human soluble LDLR, from Asp +4 to Glu +291. Therecombinant +291 form of hsLDLR, was produced in CHO cells and purifiedto homogeneity.

[0031] The immunized mice produced significant titres of specificantibodies. After screening of hybridomas, five clones (numbers 12, 28,29, 30 and 50) were identified as the highest antibody producers. Theseclones were selected for further subcloning. After subcloning, 29subclones which had high antibody productivity were isolated andampoules from the parent clones and from the subclones were frozen.

[0032] A pair of monoclonal antibodies was chosen for the ELISA to ther-hsLDLR. Mab 28 was selected as the coating antibody, and Mab 29.8,labelled with biotin, was chosen as the second antibody. Mabs 12.6 and29.8 were found to be suitable for the identification of the native andrecombinant hsLDLR in Western blot analysis and Mabs 28 and 30 werefound to be suitable for the identification of the recombinant hsLDLR inWestern blot analysis. Mabs 12.6 and 50.30 were found to be suitable forinhibiting the antiviral activity of hsLDLR.

[0033] It was also found in accordance with the invention that Mabs12.6, 28 and 29.8 inhibit replication of the viral genome of hepatitis Cvirus (HCV) in human hepatocytes primary cultures. Thus, theseantibodies may be used for the treatment of hepatitis C infection (FIG.3).

[0034] The subclass isotype of the Mab produced by the clones wasdetermined. Clones 12.6, 28, 29.8 and 30 were identified as IgG₁ whereasclone 50.30 was found to be IgM.

[0035] The Mabs, developed against the +291 form of the hsLDLRrecognised also other forms of the hsLDLR, i.e. the +292 form and the+331 form of the r-hsLDLR produced in recombinant CHO cells, in ELISAand in Western blot analysis. The +292 form comprises the N-terminuspart of the receptor from amino acid residue Asp +4 to Cys +292 and the+331 form comprises the N-terminus part of the receptor from amino acidresidue Asp +4 to Cys +331.

[0036] The antigen used to immunize mice for generating monoclonalantibodies was the r-hsLDLR +291 form, which was produced in CHO cells.Production of the r-hsLDLR was performed in bioreactors, using thestationary phase Fibracel matrix system. The r-hsLDLR was purified tohomogeneity and used for immunizing mice.

[0037] Immune spleen cells from the best mouse responder were used forfusion and generation of hybridomas.

[0038] As regards the antibodies mentioned herein throughout, the term“monoclonal antibody” is meant to include monoclonal antibodies,chimeric antibodies, fully humanized antibodies, antibodies toanti-idiotypic antibodies (anti-anti-Id antibody) that can be labeled insoluble or bound form, as well as fragments thereof provided by anyknown technique, such as, but not limited to enzymatic cleavage, peptidesynthesis or recombinant techniques.

[0039] A monoclonal antibody contains a substantially homogeneouspopulation of antibodies specific to antigens, which populationscontains substantially similar epitope binding sites. Mabs may beobtained by methods known to those skilled in the art. See, for exampleKohler and Milstein, Nature, 256:495-497 (1975); U.S. Pat. No.4,376,110; Ausubel et al., eds., Harlow and Lane ANTIBODIES: ALABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan etal., eds., Current Protocols in Immunology, Greene Publishing Assoc. andWiley Interscience N.Y., (1992-1996), the contents of which referencesare incorporated entirely herein by reference. Such antibodies may be ofany immunoglobulin class including IgG, IgM, IgE, IgA, GILD and anysubclass thereof. A hybridoma producing a mAb of the present inventionmay be cultivated in vitro, in situ or in vivo. Production of hightiters of Mabs in vivo or in situ makes this the presently preferredmethod of production.

[0040] Chimeric antibodies are molecules of which different portions arederived from different animal species, such as those having the variableregion derived from a murine Mab and a human immunoglobulin constantregion. Chimeric antibodies are primarily used to reduce immunogenicityin application and to increase yields in production, for example, wheremurine Mabs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric Mabs are used. Chimericantibodies and methods for their production are known in the art(Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984);Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984);Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., EuropeanPatent Application 125023 (published Nov. 14, 1984); Neuberger et al.,Nature 314:268-270 (1985); Taniguchi et al., European Patent Application171496 (published Feb. 19, 1985); Morrison et al., European PatentApplication 173494 (published Mar. 5, 1986); Neuberger et al., PCTApplication WO 8601533, (published Mar. 13, 1986); Kudo et al., EuropeanPatent Application 184187 (published Jun. 11, 1986); Sahagan et al., J.Immunol. 137:1066-1074 (1986); Robinson et al., International PatentApplication No. WO8702671 (published May 7, 1987); Liu et al., Proc.Natl. Acad. Sci USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad.Sci USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988);Riechmann et al., Nature 332:323-327. and Harlow and Lane, ANTIBODIES: ALABORATORY MANUAL, supra. These references are entirely incorporatedherein by reference. “Fully humanized antibodies” are moleculescontaining both the variable and constant region of the humanimmunoglobulin. Fully humanized antibodies can be potentially used fortherapeutic use, where repeated treatments are required for chronic andrelapsing diseases such as autoimmune diseases. One method for thepreparation of fully human antibodies consist of “humanization” of themouse humoral immune system, i.e. production of mouse strains able toproduce human Ig (Xenomice), by the introduction of human immunoglobulin(Ig) loci into mice in which the endogenous Ig genes have beeninactivated. The Ig loci are exceedingly complex in terms of both theirphysical structure and the gene rearrangement and expression processesrequired to ultimately produce a broad immune response. Antibodydiversity is primarily generated by combinatorial rearrangement betweendifferent V, D, and J genes present in the Ig loci. These loci alsocontain the interspersed regulatory elements, which control antibodyexpression, allelic exclusion, class switching and affinity maturation.Introduction of unrearranged human Ig transgenes into mice hasdemonstrated that the mouse recombination machinery is compatible withhuman genes. Furthermore, hybridomas secreting antigen specific hu-mAbsof various isotypes can be obtained by Xenomice immunisation withantigen.

[0041] Fully humanized antibodies and methods for their production areknown in the art (Mendez et al., Nature Genetics 15:146-156(1997);Buggemann et al., Eur. J. Immunol. 21:1323-1326 (1991); Tomizukaet al., Proc. Natl. Acad Sci. USA 97:722-727 (2000) Patent WO 98/24893.

[0042] An anti-idiotypic (anti-Id) antibody is an antibody whichrecognizes unique determinants generally associated with theantigen-binding site of an antibody. An Id antibody can be prepared byimmunizing an animal of the same species and genetic type (e.g. mousestrain) as the source of the Mab to which an anti-Id is being prepared.The immunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing an antibody tothese idiotypic determinants (the anti-Id antibody). See, for example,U.S. Pat. No. 4,699,880, which is herein entirely incorporated byreference.

[0043] The anti-Id antibody may also be used as an “immunogen” to inducean immune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original Mab, which induced the anti-Id. Thus, by using antibodiesto the idiotypic determinants of a Mab, it is possible to identify otherclones expressing antibodies of identical specificity.

[0044] Accordingly, Mabs generated against LDLR, its isoforms, analogs,fragments or derivatives of the present invention may be used to induceanti-Id antibodies in suitable animals, such as BALB/c mice. Spleencells from such immunized mice are used to produce anti-Id hybridomassecreting anti-Id Mabs. Further, the anti-Id Mabs can be coupled to acarrier such as keyhole limpet hemocyanin (KLH) and used to immunizeadditional BALB/c mice. Sera from these mice will contain anti-anti-Idantibodies that have the binding properties of the original Mab specificfor an epitope of the above LDLR protein, or analogs, fragments andderivatives thereof.

[0045] The anti-Id Mabs thus have their own idiotypic epitopes, or“idiotopes” structurally similar to the epitope being evaluated. Theterm “monoclonal antibody” is also meant to include both intactmolecules as well as fragments thereof, such as, for example, Fab andF(ab′)2, which are capable of binding antigen. Fab and F(ab′)2 fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding than anintact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

[0046] It will be appreciated that Fab and F(ab′)2 and other fragmentsof the antibodies useful in the present invention may be used for thedetection and quantitation of the LDLR protein according to the methodsdisclosed herein for intact antibody molecules. Such fragments aretypically produced by proteolytic cleavage, using enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).

[0047] A monoclonal antibody is said to be “capable of binding” amolecule if it is capable of specifically reacting with the molecule tothereby bind the molecule to the antibody. The term “epitope” is meantto refer to that portion of any molecule capable of being bound by anantibody, which can also be recognized by that antibody. Epitopes or“antigenic determinants” usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains and havespecific three dimensional structural characteristics as well asspecific charge characteristics.

[0048] An “antigen” is a molecule or a portion of a molecule capable ofbeing bound by an antibody, which antigen is additionally capable ofinducing an animal to produce antibody capable of binding to an epitopeof that antigen. An antigen may have one or more than one epitope. Thespecific reaction referred to above is meant to indicate that theantigen will react, in a highly selective manner, with an epitope on itscorresponding antibody and not with the multitude of other antibodieswhich may be evoked by other antigens.

[0049] The antibodies, including fragments of antibodies, useful in thepresent invention may be used to quantitatively or qualitatively detectthe LDLR proteins in a sample or to detect presence of cells thatexpress the LDLR proteins of the present invention. This can beaccomplished by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below) coupled with fluorescence microscopy, flowcytometric, or fluorometric detection.

[0050] The antibodies (or fragments thereof) useful in the presentinvention may be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of the LDLR proteins ofthe present invention. In situ detection may be accomplished by removinga histological specimen from a patient, and providing the labeledantibody of the present invention to such a specimen. The antibody (orfragment) is preferably provided by applying or by overlaying thelabeled antibody (or fragment) to a biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof the LDLR proteins but also its distribution on the examined tissue.Using the present invention, those of ordinary skill will readilyperceive that any of wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

[0051] Such assays for the LDLR proteins of the present inventiontypically comprises incubating a biological sample, such as a biologicalfluid, a tissue extract, freshly harvested cells such as lymphocytes orleukocytes, or cells which have been incubated in tissue culture, in thepresence of a labeled antibody capable of identifying the LDLR proteins,and detecting the antibody by any of a number of techniques well knownin the art.

[0052] The biological sample may be coupled to a solid phase support orcarrier such as nitrocellulose, or other solid support or carrier whichis capable of immobilizing cells, cell particles or soluble proteins.The support or carrier may then be washed with suitable buffers followedby treatment with a labeled antibody in accordance with the presentinvention, as noted above. The solid phase support or carrier may thenbe washed with the buffer a second time to remove unbound antibody. Theamount of bound label on said solid support or carrier may then bedetected by conventional means.

[0053] By “solid phase support”, “solid phase carrier”, “solid support”,“solid carrier”, “support” or “carrier” is intended any support orcarrier capable of binding antigen or antibodies. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon amylases, natural and modified celluloses,polyacrylamides, gabbros and magnetite. The nature of the carrier can beeither soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support or carrierconfiguration may be spherical, as in a bead, cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports or carriers include polystyrene beads. Those skilledin the art will know may other suitable carriers for binding antibody orantigen, or will be able to ascertain the same by use of routineexperimentation.

[0054] The binding activity of a given lot of antibody, of the inventionas noted above, may be determined according to well-known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

[0055] Other such steps as washing, stirring, shaking, filtering and thelike may be added to the assays as is customary or necessary for theparticular situation.

[0056] One of the ways in which an antibody in accordance with thepresent invention can be labeled is by linking the same to an enzyme andused in an enzyme immunoassay (EIA). This enzyme, in turn, when laterexposed to an appropriate substrate, will react with the substrate insuch a manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomeras, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholin-esterase. The detection can be accomplished by calorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

[0057] Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactive labeling the antibodies orantibody fragments, it is possible to detect R-PTPase through the use ofa radioimmunoassay (RIA). A good description of RIA may be found inLaboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al., North Holland Publishing Company, NY (1978) with particularreference to the chapter entitled “An Introduction to Radioimmune Assayand Related Techniques” by Chard, T., incorporated by reference herein.The radioactive isotope can be detected by such means as the use of a gcounter or a scintillation counter or by autoradiography.

[0058] It is also possible to label an antibody in accordance with thepresent invention with a fluorescent compound. When the fluorescentlylabeled antibody is exposed to light of the proper wavelength, itspresence can be then detected due to fluorescence. Among the mostcommonly used fluorescent labeling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrine, pycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine.

[0059] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²E, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriamine pentaacetic acid (ETPA).

[0060] The antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0061] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0062] An antibody molecule of the present invention may be adapted forutilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier and a quantity of detectably labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody.

[0063] Typical, and preferred, immunometric assays include “forward”assays in which the antibody bound to the solid phase is first contactedwith the sample being tested to extract the antigen from the sample byformation of a binary solid phase antibody-antigen complex. After asuitable incubation period, the solid support or carrier is washed toremove the residue of the fluid sample, including unreacted antigen, ifany, and then contacted with the solution containing an unknown quantityof labeled antibody (which functions as a “reporter molecule”).

[0064] After a second incubation period to permit the labeled antibodyto complex with the antigen bound to the solid support or carrierthrough the unlabeled antibody, the solid support or carrier is washed asecond time to remove the unreacted labeled antibody.

[0065] In another type of “sandwich” assay, which may also be usefulwith the antigens of the present invention, the so-called “simultaneous”and “reverse” assays are used. A simultaneous assay involves a singleincubation step as the antibody bound to the solid support or carrierand labeled antibody are both added to the sample being tested at thesame time. After the incubation is completed, the solid support orcarrier is washed to remove the residue of fluid sample and uncomplexedlabeled antibody. The presence of labeled antibody associated with thesolid support or carrier is then determined, as it would be in aconventional “forward” sandwich assay.

[0066] In the “reverse” assay, stepwise addition first of a solution oflabeled antibody to the fluid sample followed by the addition ofunlabeled antibody bound to a solid support or carrier after a suitableincubation period is utilized. After a second incubation, the solidphase is washed in conventional fashion to free it of the residue of thesample being tested and the solution of unreacted labeled antibody. Thedetermination of labeled antibody associated with a solid support orcarrier is then determined as in the “simultaneous” and “forward”assays.

[0067] The invention will be now illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Preparation of CHO r-hsLDLR

[0068] Stable recombinant CHO cells expressing human soluble LDLR weregenerated by co-transfection of CHO-DUKX cells lacking the dihydrofolatereductase (DHFR) gene (Urlaub, G. et al., 1980) with two expressionvectors: psLDLR01 containing the N-terminal ligand-binding domain of theLDLR, beginning at amino acid residue Asp (+4) up to Glu 291 (+291), andpDHFR, containing the murine gene for DHFR, both controlled by thepromoter and transcription termination elements of the SV40 earlyregion. Transfection was performed by cationic liposomes usingLipofectAmine (Gibco BRL), according to the protocol described by themanufacturer. Seventy-two hours after transfection cells weretransferred to a selective medium lacking deoxy and ribonucleosides andsupplemented with 10% dialysed FCS. Cells expressing DHFR activity wereable to form colonies, which were isolated by lifting the cells withtrypsin-soaked paper discs The cells were grown and screened forr-hsLDLR activity. The transfected cells were then subjected to geneamplification by MTX, followed by subcloning and selection of the stableproducer clones. r-hsLDLR (+291 form) was produced with cells of astable CHO producer clone designated #33-10-29-21, in a 5 liter CelliGenbioreactor in serum free medium (Gibco CHO-A-SFM Cat. no. 95-0091DJ).The crude harvest was clarified by filtration through a 0.8-02 pcartridge filter (Gelman Cat. No. CSS92DSCCK) and concentrated 100 foldover a 5-kDa membrane. The +291 form of the r-hsLDLR used for the firstimmunizations, was purified using a small scale purification process. Inthis process a DEAE-Sepharose cation exchange column was used, followedby a hydrophobic interaction step on a Butyl-TSK column followed by anHTP column and a size exclusion chromatography (SEC) step on a Sephacryl100 column. Fraction #27 of the SEC, was chosen, as it contained aspecific antiviral activity of 780 units/μg detected in the antiviralassay described in Example 9 below. The protein in this fraction wasidentified as r-hsLDLR by N-terminal analysis.

[0069] A second batch, of CHO +291 r-hsLDLR was purified and used forboost injections of the mice. It was purified using a refined processhaving an improved yield which included the following steps: a)clarification and concentration x100 of the crude harvest; b) a HQ POROSanion exchange column, and c) two hydrophobic interaction (HIC) steps:capture on a Butyl-TSK column and flow through a Phenyl 5PW column. Theunbound fraction from the last HIC step was dialysed and purified overan HS-POROS cation exchange column. The last step was a Hydroxyapatite(HTP) column. The r-hsLDLR hence obtained was purified to about 90%,eluting as a single major peak in RP-HPLC.

Example 2 Immunization of Mice

[0070] b 10 μg of the purified r-hsLDLR of fraction #27 of the SECcolumn of Example 1 above, at a concentration of 100 μg/ml, werehomogenised with Complete Freund's Adjuvant (CFA, 50% v/v) and injectedinto the footpad of each of five 7 week old Balb/C female mice.

[0071] Four weeks after the first immunization, the mice were boosted,intramuscular with 10 μg of the same fraction of purified r-hsLDLR, in a50% (v/v) solution of CFA.

[0072] Two weeks after the second injection the mice sera was tested forantibodies to r-hsLDLR, using the direct ELISA described in Example 3below.

[0073] The two mice M-1 and M-2, with the most significant specificimmunoreactivity with r-hsLDLR were further boosted, 10 weeks after thesecond injection, with 10 μg of the purified r-hsLDLR obtained in therefined purification process described in Example 1 above.

[0074] The mice were bled 14 weeks later and tested for antibodies tor-hsLDLR. They were then given two additional boosts of 50 μg r-hsLDLRin PBS: the first intraperitoneal and the second, two days later, bothintraperitoneal and intravenous.

[0075] The mice were bled two weeks after the second injection and theantiserum was tested for anti-r-hsLDLR activity by the direct ELISA ofExample 3 below. Each antiserum was serially diluted 1:100-1:32,000 andapplied in duplicates to a 96 well plate coated with 10U/well ofr-hsLDLR purified using the refined purification process described inabove Example 1. Assay buffer and DMEM +10% HS containing PBS +1% BSA orGelatin +0.05% Tween 20+0.05% Thimerosal were used as blanks in thefirst well of each row. Normal Mouse Serum (NMS) was applied in the samedilution range in the last two rows as negative controls. The absorbencyof the enzymatic reaction was measured with an ELISA reader at 492 and405 nm.

[0076] The results of this test indicated that sera from mouse M-l had ahigher specific immunoreactivity with r-hsLDLR and was thereforesacrificed and spleen cells were collected for fusion with myeloma cells(Eshhar Z, 1985).

Example 3 Direct ELISA for Antisera Testing and Hybridoma ClonesScreening

[0077] The direct ELISA for screening for positive antisera wasperformed as following: 96 wells plates were coated with 100 μl ofr-hsLDLR (purified by the refined purification process of example 1) 100units/ml (10U/well) in PBS +1% Gelatine (Sigma, Cat. No. G-7765)+0.9 mMCa⁺² and 0.5 mM Mg⁺², pH 5.6, hereinafter referred to as assay buffer,for 90 min. at 37° C. with shaking. The plates were washed six times inPBS +0.05% Tween 20 (Polyoxyethylene-Sorbitan Monolaurate-Sigma P-1379),hereinafter referred to as wash solution.

[0078] Anti serum samples from the immunized mice serially diluted1:100-1:32,000, or supernatant from hybridoma cell cultures were addedto the wells and incubated for 90 min. at 37° C., while shaking,followed by six washes in wash solution.

[0079] 100 μl of Horse Radish Peroxidase (HRP)-APA conjugated goatantibody to mouse Fab (Sigma-Cat. NO. 4601-1) diluted 1:1,200 were addedto the wells and incubated for 90 min at 37° C., while shaking, and thenwashed six times with wash solution.

[0080] 100 μl of substrate solution (prepared by dissolving one tabletof OPD and one tablet of H₂O₂ in 20 ml water) were added to the wellsand incubated at RT for 30 min. The enzymatic reaction was stopped bythe addition of 100 μl/well of 4N HCl.

[0081] The absorbency in the 96 wells plates was read using an ELISAreader at 492 and 405 nm and the results were calculated using the fourparametric logistic algorithm, by the MultiCalc software of the PCcomputer linked to the ELISA reader.

Example 4 Fusion, Hybridoma Preparation, Selection of Clones andPurification of Antibodies from Ascitis Fluids

[0082] The fusion process and hybridoma cell selection were performedaccording to the protocols in Eshhar Z, 1985. Briefly, spleen cells frommouse M-1, boosted 2-4 days before fusion, were fused with myeloma cellsby a short incubation with PEG. The PEG was first slowly diluted withDMEM and then completely removed by centrifugation. The cells werere-suspended in DMEM-HAT medium, distributed in 96 wells plates at aconcentration of about 3.4×10⁻⁴ cells/well and incubated for 10-14 daysin an 8% CO₂ incubator at 37° C. The medium in all the hybridoma wellswas changed to DMEM supplemented with 10% Horse Serum (HS) within 10days. Hybridoma culture supernatant samples were screened for thepresence of Mabs to r-hsLDLR by the direct ELISA described in Example 3above. Assay buffer and DMEM+10% HS were used as blanks, Mab C7(commercially available from Amersham) and M-1 mouse antiserum were usedas positive controls, while a monoclonal antibody to the soluble p55 TNFreceptor was used as a negative control. Cells from wells, in which thepresence of antibodies was detected in the culture supernatant, weretransferred to 24 well plates and then to 25 cm² T-flasks. The expandedcultures were monitored for secretion of Mabs to r-hsLDLR. Ampoules ofcells from positive cultures were frozen and stored in liquid nitrogen.

[0083] A total of approximately 1000 cultures were screened fordetecting antibodies to r-hsLDLR. 54 cultures with the highestimmuno-activity were re-tested several times. Five cultures (12, 28, 29,30 and 50) with the highest activity were cloned by limiting dilution in96 well plates. Supernatants from the growing clones were tested severaltimes for antibodies to r-hsLDLR, by the direct ELISA.

[0084] Cells of positive hybridoma clones were grown in tissue cultureflasks in DMEM containing 15% horse serum and ampoules were frozen frompart of the cultures. In parallel, cells of different hybridoma cloneswere injected, to 2-4 mice each, to obtain ascitis fluids. Antibodieswere purified from ascitis fluid either by ammonium sulphateprecipitation or on a protein G column. Briefly 7.5 ml of ascitis fluidwere diluted 1:3 in 20 mM Phosphate buffer pH 7 and loaded onto a 5 mlProtein G column (C10/10). The column was washed with 20 mM Phosphatebuffer pH 7 and the Mabs were eluted with 100 mM Glycine buffer pH 2.7.The pH of the elution fraction was adjusted to 7-7.5 with IM Tris bufferpH 9.3.

Example 5 Screening for Pairs of Mabs to be used in ELISA andOptimisation of the ELISA Parameters

[0085] The Mabs purified from the ascitis fluids as in example 4 abovewere used to perform a set of experiments in a matrix format to selectthe best suitable pair of Mabs to be used as first and second antibodiesin the sandwich ELISA for r-hsLDLR described in Example 6 below.Briefly, 96 well plates were coated with ascitis fluids derived fromfive hybridomas (# 12, 28.28, 29.08, 30 and 50.05) purified either byammonium sulphate precipitation or on a protein G column. The antibodieswere screened using the +291 form as well as the +292 (from amino acidresidue Asp +4 to Cys +292) and +331 (from amino acid residue Asp +4 toCys +331) forms of r-hsLDLR produced in CHO cells, as antigens. One mlof each of the above partially purified Mabs was labelled with biotinfor a fast screening of their suitability as second antibodies in asandwich ELISA. Briefly 1.5 mg of ammonium sulphateprecipitation-purified Mabs were adjusted to pH 8.5 with 30 μl of 0.5 MNaHCO₃. 0.75 mg of Biotin-OSu N-Hydroxysuccinimido-Biotin (Biotin-OSu,Sigma, Cat. # H1759, from a solution of 5 mg in 200 μl DMSO) were addedto the antibody solution and incubated for two hours at room temperaturewith gentle shaking, followed by an overnight incubation at 2-8° C. Thereaction solution was loaded onto a Sephadex G-25M (Pharmacia Cat.#17-0851-01) PD10 column to separate between the biotinylated Mabs andthe excess of non-reacted biotin-OSu.

[0086] The first preliminary experiments indicated that Mabs 29.08 and30 produced the highest signal above background, when used as secondantibodies in the ELISA.

[0087] The reaction of these two clones was tested again as secondantibodies with antibodies 12, 28, 29.08 and 50 used for coating ofplates. The results of this experiment clearly showed that Mab 28 wasthe antibody most suitable for coating.

[0088] The best results, in terms of signal intensity and specificity,were obtained with Mab 28 used for coating of the microtiter plate, andMab 29.08, labelled with biotin, as a second antibody. Using these Mabsgood results were obtained with all the three forms (+291, +292 and+331) of the r-hsLDLR. With all forms, the absorbency at 492/405 nm wasabout 1.3 OD.

[0089] The three forms of the r-hsLDLR antigen were analysed in serialdilutions in a concentration range of 0.9-1000 ng/ml. A dose responsecurve was obtained with Mab 28 used for coating and biotinylated Mab29.08 as a second antibody. This combination gave a linear response at aconcentration range of 1-10 ng/ml of r-hsLDLR.

[0090] The various parameters that may affect the ELISA test such asconcentration of reagents, incubation periods, selection of buffers andplates were optimised by testing the following parameters:

[0091] Coating of microtiter plate wells with 5-10 μg/ml of Mab28 inPBS.

[0092] F Buffer composition:

[0093] a) PBS+Tween 20

[0094] b) Tris+Ca⁺²+NaCl+Tween 20

[0095] Blocking solutions:

[0096] a) 1% Gelatine in PBS, 0.05% Tween, 0.005% Thimerosal

[0097] b) 1% BSA in PBS, 0.05% Tween, 0.005% Thimerosal

[0098] c) 1% FBS in PBS, 0.05% Tween, 0.005% Thimerosal

[0099] d) 1% Milk in PBS, 0.05% Tween, 0.005% Thimerosal

[0100] e) I Block, Hy Pep and Hy Yeast

[0101] Second Mab 29.08, labelled with biotin, at concentrations of1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:10,000 equivalent to aconcentration range of 10.74-0.537 μg/ml.

[0102] Extravidin concentrations: 1:500, 1:1000, 1:2000, 1:4000, 1:8000,1:10,000 equivalent to a concentration range of 4-0.2 μg/ml.

[0103] On the basis of these experiments the final procedure for thesandwich ELISA test described in Example 6 below was established.

Example 6 Establishment of a Sandwich ELISA for r-hsLDLR

[0104] A sandwich ELISA to the r-hsLDLR was established using Mabs 28and 29.08. Briefly 96 wells plate were coated with 100 μl of a Protein Gpurified Mab 28 (5 μg/ml) overnight at 2-8° C. or 3 hrs. at 37° C. Theplates were then washed five times with PBS+0.05% Tween 20. The plateswere incubated with 200 μl of blocking solution (PBS+1%BSA or Gelatin+0.05% Tween 20+Thimerosal 0.05% for one hr at 37° C. or over night at4° C. and washed five times with PBS+0.05% Tween 20. 100 μl of samplesor of calibration curve antigen (CHO +291 r-hsLDLR, 0.5-32 ng/ml dilutedin blocking solution), were added to the wells and incubated for 90 minat 37° C., with shaking. The plates were then washed five times withPBS+0.05% Tween 20.

[0105] 100 μl/well biotinylated Mab 29.08 (0.67 μg/ml) in blockingsolution were added, and incubated with shaking for one hour at 37° C.The plates were washed five times with PBS+0.05% Tween 20. 100 μl of acommercial extravidin—peroxidase conjugate, (ExtrAvidin TM-PeroxidaseBioMakor, Cat.# 0645-1) diluted 1:10,000 were added to the wells andincubated with shaking for one hour at 37° C. The plates were thenwashed five times with PBS+0.05% Tween 20. 125 μl of the abovementionedsubstrate solution were added to each well and incubated for about 10minutes until the colour developed to the desired intensity. Thereaction was stopped by adding 125 μl of 4N HCL. The absorbency in the96 wells plates was read using an ELISA reader at 492 and 405 nmwavelengths and the results were calculated by the MultiCalc software ofthe PC computer linked to the ELISA reader.

Example 7 Monoclonal Antibodies Isotype

[0106] The monoclonal antibodies Ig isotype was determined using acommercial isotyping kit (PharMingen International) according to themanufacturer's assay procedure. Clones 12.6, 28, 29.8 and 30 wereidentified as IgG₁, whereas clone 50.30 was found to be of the IgMclass.

Example 8 SDS-PAGE Western Blot Analysis

[0107] The +291 form of purified r-hsLDLR and the native LDLR purifiedfrom human urine were analysed by western blot analysis with themonoclonal antibodies developed to the r-hsLDLR. Briefly a 12% SDS PolyAcrylamide gel was loaded with 100 ng/lane of the CHO +291 form ofr-hsLDLR, or native urinary hsLDLR or TBP-1 crude harvest (as negativecontrol) under reducing conditions (40 mM DTT). One lane was loaded withLow Molecular Weight Markers (LMW). This set of samples was run fivetimes. The proteins separated on the gels were transferred byelectroelution to nitrocellulose membranes. The membranes were incubatedin PBS containing 10% low-fat milk, 0. 1% Tween 20, for 16 hr. Themembranes were cut into strips and each strip was incubated for 2 hoursat room temperature with one of the five selected Mabs: 12.6, 30, 50.30,28 or 29.08 (ascitis fluid diluted 1:4000).

[0108] Membrane strips were washed with PBS containing 0.1% Tween 20(3×15 min) and incubated for one hour with the second antibody—goatanti-mouse conjugated to horseradish peroxidase-alkaline phosphatase(diluted 1:10.000, BioMakor) for 2 hours at room temperature.

[0109] The strips were washed with PBS containing 0.1% Tween 20 (3×15min). The positive bands were detected by enhanced chemiluminescence(ECL, Amersham).

[0110] Monoclonal antibodies #12.6 and #29.8 recognised both the urinaryas well as the +291 form of the purified r-hsLDLR in western blotanalysis (FIG. 2). Mabs 28 and 30 recognised the +291 form of thepurified r-hsLDLR.

Example 9 Inhibition of r-hsLDLR Antiviral Activity by MonoclonalAntibodies

[0111] Mabs which specifically reacted with r-hsLDLR were tested fortheir ability to block the antiviral activity of r-hsLDLR (+291 form)in-vitro, using a cytopathic effect (CPE) inhibition assay in a VSV/WISHsystem.

[0112] WISH cells (of human amnion origin) were cultured in MEMsupplemented with 10% FBS and 4 mM glutamine in a 37° C., 5% CO₂incubator. Exponentially growing cells were seeded in 96-well tissueculture plates, at a density of 40,000 cells/well twenty-four hoursbefore initiation of the assay. Samples to be tested and the standardwere diluted and dispensed into the cells' containing wells. VSV wasimmediately added to the wells, at a multiplicity of Infection (MOI) of0.5 pfu/cell. The plates were incubated 16-18 hours at 37° C. and werethen washed with ethanol. The monolayer of surviving cells was viewed byGram Crystal Violet stain. Quantitation of the cytopathic effectrelative to the standard was performed by plotting the colour densityversus standard concentration.

[0113] For analysing the neutralising effect of the antibodies, r-hsLDLRwas pre-incubated for 30 min. at 37° C., with increasing concentrationsof ascitis fluid of the Mab tested. These solutions were then added tocultures of WISH cells in 96 microtiter plates, followed by the additionof vesicular stomatitis virus (VSV). After 18 hours incubation, the VSVmediated cell lysis was determined by staining of the remaining cellswith crystal violet. Semi-quantitation of the cytopathic effect relativeto the standard was performed by plotting the colour intensity(determined by an ELISA reader) versus standard concentration.

[0114] The effect of the Mabs was tested with increasing concentrationsof r-hsLDLR. As shown in Table 1 two Mabs (12.6 and 50.30) were found todisplay neutralising activity.

[0115] In the experiment shown in Table 1, the inhibitory effect of thetwo Mabs was tested at a 1:40 dilution of the ascitis fluids. At thisdilution, Mab 12.6 displayed somewhat higher activity than to Mab 50.30.This may result from the properties of the Mabs, as well as fromdifferences in their concentration in the ascitis fluid.

[0116] The inhibitory effect of the Mabs could be overcome by increasingr-hsLDLR relative to Mabs concentration. In fact at r-hsLDLRconcentrations of 62.5 U/ml, neither Mab had any effect on r-hsLDLRactivity at the antibody concentration analysed. TABLE 1 Inhibition ofr-hsLDLR antiviral activity by clones 12.6 and 50.30¹ LDLR Concentration(U/ml) VSV/Mab 0 2.5 12.5 62.5 −VSV 1.5 1.2 1.6 1.5 +VSV (0.25) 1 7(1.7) 1.7 +VSV + Clone 50.30 6 (0.4) 0.88 (1.25) 1.62 +VSV + Clone 12.65 (0.5) 0.77 7 (0.7) 1.67 # Mabs was determined at a 1:40 dilution ofthe ascitis fluid. The number of viable cells is represented in thetable in OD values. Numbers # in brackets represent a repetitionperformed at the 0 and 12.5 U/ml LDLR concentrations.

[0117] Inhibition of the antiviral activity of r-hsLDLR was determinedusing increasing concentrations of Mabs 12.6 and 50.30. Mab 12.6,inhibited the antiviral activity of r-hsLDLR, by ˜60% at a 1:40 dilution(of the ascitis fluid) and by ˜35% at a 1:20,500 dilution. Clone 50.30inhibited r-hsLDLR activity by ˜45% at the 1:40 dilution and by ˜15% at1:20,500 dilution.

[0118] The dose response curve, obtained with both Mabs, and theobservation that their inhibitory effect was impaired by excessr-hsLDLR, suggest that the Mabs exert their effect by binding tor-hsLDLR.

Example 10 Inhibition of HCV Replication by Monoclonal Antibodies

[0119] Mabs specific for r-hsLDLR were tested for their ability toinhibit HCV replication in human hepatocytes in primary culture. FT167cell culture was derived from a 57 year old male patient requiringlobectomy resection for medical purposes (metastasis of a colon tumor,right lobe).

[0120] Primary cultures of human hepatocytes were prepared by the twosteps collagenase perfusion method (Maurel P. Adv.Drug Del.Rev.22:105-132 (1996), Pichard L. et al. Mol. Pharmacol. 41:1047-1055(1992),Ferrini JB. Et al. Chem-Biol Interactions 107:31-45 (1997)). Theviability of the cells before plating was determined using trypan blueexclusion test. Four million cells in 3 ml of culture medium were placedinto 60-mm plastic dishes precoated with collagen The long-termserum-free culture medium consisted of Williams'E, supplemented aspublished (Lanford R. et al. In Vitro Cell Dev. Biol.25:174-182 (1989)).This medium was subsequently renewed every 48 hours. Cultures weremaintained at 37° C. in a humid atmosphere of air and 5% carbon dioxide.Under these culture conditions, human hepatocytes retain theirdifferentiated phenotype for at least 35 days (Ferrini J B. Et al.Chem-Biol Interactions 107:31-45 (1997)) and are sensitive to HCVInfection and permissive to the viral genome replication (Fournier C. etal. J. Gen Virol. 79:2367-2374 (1998)).

[0121] HCV-positive serum sample: A bank of human sera from patientstested anti-HCV antibody-positive by the EIA HCV 3.0 and Chiron RIBA HCV3.0 SIA has been established. None of these patients was co-Infectedwith HBV or HIV. In each serum sample HCV RNA was quantitated by theRoche monitor and genotyped by a line probe assay (Inno-Lipa HCV II,Innogenetics). Serum samples were stored at −80° C., in small aliquotsin order to avoid freezing-thawing cycles. In these experiments thesample S42 (genotype 1b; viral load: 410 000 copies/ml) was used.

[0122] For infection and subsequent treatments, hepatocyte cultures weretransferred under sterile conditions to a P3-laboratory (highconfinement for human-IFNectious micro-organisms). Three days afterplating, when the cells had recovered from the traumatism of isolation,in vitro infection of hepatocytes was performed by an overnightincubation with 25 μL of HCV-positive serum sample (S42) in 3 mL ofmedium. After infection, cells were washed three times with 3 mL offresh medium and the culture was continued under normal conditions inthe long-term culture medium.

[0123] Cells were treated with 3 different Mabs against r-hsLDLR,Mab12.6, Mab28 and Mab29.8. Thirty minutes before infection, cells wereexposed to 2 or 8 μg/ml of the different Mabs. Then cells were infectedas described above.

[0124] Control cultures were infected under similar conditions but inthe absence of antiviral treatment. In parallel experiments, the samecultures were treated with 5000 U/mL IFNα, under similar conditions forcomparison (IFNα strongly inhibits HCV replication in the cells ref).All treatments were carried out in duplicate.

[0125] At day 5 post-Infection, the medium was removed and the cultureswashed 3 times with cold phosphate-buffered saline. RNA was purifiedfrom 4×10⁶ hepatocytes using a guanidinium isothiocyanate-acid phenolextraction procedure (Chomczynski P N. And Sacchi N. Analyt. Biochem.162:156-159 (1987)). The precipitated RNA was dissolved in 50 μL ofdiethylpyrocarbonate (DEPC)-treated water and quantified. One μg ofcellular RNA was analyzed in the strand-specific rTth RT-PCR assay.

[0126] To avoid possible contamination, the strand-specific RT-PCR assaywas carried out sequentially using three different rooms: a pre-PCRroom, a PCR room and a post-PCR room. RNA dissolved in 10 μl ofDEPC-treated water was covered with mineral oil and heated at 95° C. for1 min. The temperature was lowered to 70° C. and 10 μl of preheated cDNAreaction mixture was added. The temperature was then dropped to 60° C.for 2 min for annealing and the cDNA reaction was performed for 20 minat 70° C. using the rTth DNA polymerase (Perkin-Elmer). The temperaturewas maintained at 70° C. while 40 μl of prewarmed buffer containing EGTAas chelator of Mn²⁺ was added to suppress the rTth RT activity. Reactiontubes were held at 70° C. while 40 μl of prewarmed PCR mixture wasadded. The PCR conditions, performed on Gene Amp ® PCR-System 9600(Perkin-Elmer), consisted of an initial cycle at 94° C. for 1 min, 50cycles at 94° C. for 15 sec, 58° C. for 30 sec, 72° C. for 30 sec and afinal extension step at 72° C. for 7 min. For positive strand HCV RNAassay, the nucleotide sequence of the reverse primer P3 is:5′-TGG/ATGCACGGTCTACGAGACCTC-3′, (nt 342-320) and that of the forwardprimer P4 is: 5′-CACTCCCCTGTGAGGAACT-3′, (nt: 38-56), (Laskus T. et al.J. Gen. Virol. 78:2747-2750 (1997)). The same primers were used inreverse order to detect the negative strand. One-tenth of the amplifiedproduct was analyzed by gel electrophoresis on agarose (2%), followed bycoloration with BET and photography under UV light. In all series ofexperiments, dilutions of synthetic HCV RNA (+) and (−) strands weremade and 1 μg of total liver RNA was added to mimic the conditions foranalysis of cultured hepatocytes. These mixtures were used as positivecontrols for RT-PCR assay and analysis.

[0127]FIG. 3 shows that the production of the HCV negative strand, inthe precence of the Mabs againt LDLr, was fully inhibited in cultureFT167. Therefore the replication of the viral genome was stronglyinhibited. The results were consistent with the view that the LDLR mightbe a receptor for HCV.

Example 11 Production of Chimeric Antibodies to LDLR

[0128] mRNA is purified from a hybridoma line producing mAb specific forLDLR.

[0129] Specific cDNA is synthesized with olygonucleotides complementaryto the 5∝ end of the exon CH1 from the heavy chain variable domain(oligo 1) and from the 5′ of the Cκhexon of the light chain variabledomain (oligo 2) using the purified mRNA as the template.

[0130] Two cDNAs are obtained one of which encode the variable region(specific for LDLR) of the heavy chain, and the other the variableregion (specific for the LDLR) of the light chain. The cDNAs are clonedand sequenced.

[0131] For the construction of the chimeric heavy chain, the variableregion of a cloned human Ig heavy chain gene is exchanged (using geneticmanipulations) for the cloned DNA encoding the mouse variable domain(specific for LDLR) of the heavy chain. The genetic manipulationsinclude excition of the variable region from the human Ig, usingspecific restriction enzymes and ligation of the mouse variable region.The same procedure is performed to obtain the chimeric light chain.

[0132] Two mammalian expression plasmids are constructed, one includingthe chimeric heavy chain gene and the other including the chimeric lightchain gene. Both vectors are used to cotransfect the hybridoma cell line(SP6).

[0133] The production of LDLR specific Ig is tested by ELISA or westernblots using culture soup of transfectant cells as secondary antibody.The affinity of the chimeric antibody to its ligand is monitored byBiacore.

Example 12 Preparation of Transgenic Mice that are Engineered to ContainHuman Immunoglobulin Gene Loci (Xenomice) and Preparation of Human mAbagainst hLDLR.

[0134] Xenomice preparation is described in WO 98/24893 and Mendez M, J.et al Nature genetics 15:146-56(1997).

[0135] Human-yeast artificial chromosome (YAC) libraries are screenedfor YACs containing the human heavy chain variable region (about 1000kb) (YAC cloning method is the method of choice when inserts sizesbigger of 100 kb are required).

[0136] The YACs are characterized by Southern blot analysis and by PulseField Electrophoresis (PFGE). The YACs should include the Cμ Cδ, Dh andVh regions in germ line configuration.

[0137] Through utilization of the overlapping sequences contained in theYACs, the YACs are recombined in yeast by stepwise recombinationstrategy. Prior to recombination the 3′ end YAC (with the V region) isligated to HPRT selectable marker. The structure of recombined YAC isconfirmed by PFGE and Southern blot analysis (presence of the humanheavy chain locus from C region to Vh region in germline configuration).

[0138] The YAC acentric arm is targeted with a vector bearing thecomplete y2 constant region, mouse enhancer, neomycin resistance gene,to yield the final heavy chain containing the whole variable region i.e.82 Vh genes, 6 Jh genes and 3 different constant regions Cμ Cδ Cγ withtheir corresponding regulatory sequences. This YAC is designated yH2.This construct is used for the production of the Xenomouse.

[0139] A similar strategy to the one used above is utilized for thereconstruction of the kappa loci, only that: neomycin selection markeris ligated to the reconstructed YAC containing the whole kappa loci.This YAC is designated yK2.

[0140] YACs containing the yH2 are introduced into ES cell via fusion ofYAC containing yeast spheroplast with HPRT deficient E14.TG3B mouse EScells. HPRT positive cells are selected. Positive clones are propagatedand analyzed by Southern blots and by CHEF blot analysis. Clonescontaining the intact yH2 YAC are selected.

[0141] Introduction and selection of yK2 YAC in ES cells is performedsimilarly as described for yH2 YAC.

[0142] YH2 containing ES cells are microinjected into mouse C57BL/6Jblastocytes. The chimeric males produced are evaluated for germ linetransmission to offspring.

[0143] yH2 and or yK2-transgenic mice are bred with DI mice (homozygousfor gene targeted-inactivated mouse heavy and kappa chain loci). Each ofthe yH2;DI transgenic strains are bred with yK2;DI transgenic strain togenerate Xenomouse strains.

[0144] Reconstitution of B-cell development and antibody production inXenomouse is evaluated by flow cytometry and ELISA.

[0145] The immunization of xenomouse is performed as described inexample 2.

[0146] The methods for the hybridoma preparation and screening ofpositive clones are similar to those described in examples 3 and 4.

[0147] Hybridoma clones 12.6, 28, 29.8, 30 and 50.30 were deposited atthe Collection Nationale de Culture de Microorganismes (CNCM), InstitutPasteur, Paris, under the Budapest Treaty and were accorded deposit Nos.1-2390, 1-2391, 1-2392, 1-2393 and 1-2394, respectively.

REFERENCES

[0148] Agnello, V., Abel, G., Elfahal, M., Knight, G. B., and Zhang, Q.X. (1999). “lepatitis C virus and other flaviviridae viruses enter cellsvia low density lipoprotein receptor [In Process Citation].” Proc NatlAcad Sci U S A, 96(22), 12766-71.

[0149] Beisiegel, U., Schneider, W. J., Goldstein, J. L., Anderson, R.G., and Brown, M. S. (1981). “Monoclonal antibodies to the low densitylipoprotein receptor as probes for study of receptor-mediatedendocytosis and the genetics of familial hypercholesterolemia.” J BiolChem, 256(22), 11923-31.

[0150] Bieri, S., Djordjevic, J. T., Daly, N. L., Smith, R., and Kroon,P. A. (1995). “Disulfide bridges of a cysteine-rich repeat of the LDLreceptor ligand-binding domain.” Biochemistry, 34(40), 13059-65.

[0151] Brown, M. S., and Goldstein, J. L. (1976). “Familialhypercholesterolemia: A genetic defect in the low-density lipoproteinreceptor.” N Engl J Med, 294(25), 1386-90.

[0152] Brown, M. S., and Goldstein, J. L. (1986). “A receptor-mediatedpathway for cholesterol homeostasis.” Science, 232(4746), 34-47.

[0153] Chomczynski, P. N., And Sacchi, N. (1987). “Single-step method ofRNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction”. Analyt. Biochem. 162:156-9.

[0154] Eshhar Z, 1985 “Monoclonal Antibody Strategy and Techniques” in“Hybridoma technology in the bioscience and medicine”, Edited by TimothyA. Springer (Plenum Publishing Corporation, 1985; Chapter 1)

[0155] Fischer, D. G., Tal, N., Novick, D., Barak, S., and Rubinstein,M. (1993). “An antiviral soluble form of the LDL receptor induced byinterferon”. Science, 262(5131), 250-3.

[0156] Fischer, D. G., Novick, D., Cohen, B, Rubinstein, M. (1994).“Isolation and characterization of a soluble form of the LDL receptor,an interferon-induced antiviral protein”. Proc Soc Exp Biol Med206(3),228-32.

[0157] Ferrini, J. B., Pichard, L., Domergue, J., and Maurel, P. (1997).“Long-term primary cultures of adult human hepatocytes”. Chem-BiolInteractions 107:31-45.

[0158] Fournier, C., Sureau, C., Coste, J., Ducos, J., Pageaux, G.,Larrey, D., Domergue, J., and Maurel, P. (1998). “In vitro infection ofadult normal human hepatocytes in primary culture by hepatitis C virus”.J. Gen Virol. 79:2367-74.

[0159] Goldstein, J. L., Anderson, R. G., and Brown, M. S. (1979).“Coated pits, coated vesicles, and receptor-mediated endocytosis.”Nature, 279(5715), 679-85.

[0160] Goldstein, J. L., Dana, S. E., Brunschede, G. Y., and Brown, M.S. (1975). “Genetic heterogeneity in familial hypercholesterolemia:evidence for two different mutations affecting functions of low-densitylipoprotein receptor.” Proc Natl Acad Sci USA, 72(3), 1092-6.

[0161] Lanford R. E., Carey, K. D., Estlack, L. E., Smith, G. C., andHay, R. V. (1989) “Analysis of plasma protein and lipoprotein synthesisin long-term primary cultures of baboon hepatocytes maintained inserum-free medium” In Vitro Cell Dev. Biol. 25:174-82.

[0162] Laskus T., Radkowski, M., Wang, L. F., Cianciara, J., Vargas, H.,and Rakela, J. (1997). “Hepatitis C virus negative strand RNA is notdetected in peripheral blood mononuclear cells and viral sequences areidentical to those in serum: a case against extrahepatic replication”.J. Gen. Virol. 78:2747-50.

[0163] Maurel P. (1996) “The use of adult human hepatocytes in primaryculture and other in vitro systems to investigate drug metabolism inman”. Adv.Drug Del.Rev. 22:105-132

[0164] Mendez, M. M., Green, L. L., Corvalan, J. R. F., Jia X-C.,Maynard-Currie, E. E., Yang, X-D., Gallo, M. L., Louie, D. M., Lee, D.V., Erickson, K. L., Luna, J., Roy, C. M-N., Abderrahim, H.,Kirshenbaum, F., Noguchi, M., Smith, D. M., Fukushima, A., Hales, J. F.,Finer, M. H., Davis, C. G., Zsebo, K. M. and Jakobovits, A. (1997).“Functional transplant of megabase human immunoglobulin locirecapitulates human antibody response in mice”. Nature Genetics, 15,146-56.

[0165] Pichard, L., Fabre, I., Daujat, M., Domergue, J., Joyeux, H., andMaurel, P. (1992). “Effect of corticosteroids on the expression ofcytochromes P450 and on cyclosporin A oxidase activity in primarycultures of human hepatocytes”.Mol. Pharmacol. 41:1047-55.

[0166] Riachmann, L., Clark, M., Waldmann, H., and Winter, G. (1988).“Reshaping human antibodies for therapy.” Nature, 332, 323-27.

[0167] Sudhof, T. C., Goldstein, J. L., Brown, M. S., and Russell, D. W.(1985). “The LDL receptor gene: a mosaic of exons shared with differentproteins.” Science, 228(4701), 815-22.

[0168] Urlaub, G. and Chasin, L. A. (1980) Isolation of Chinese HamsterCell Mutants Deficient in Dihydrofolate Reductase Activity. Proc. Natl.Acad. Sci. USA 77: 4216-4220.

1. A monoclonal antibody, chimeric antibody, fully humanized antibody,anti-anti-ID antibody or fragment thereof which specifically recognizesand binds human LDL receptor and fragments thereof, except monoclonalantibody C7.
 2. A monoclonal antibody according to claim 1 whichspecifically recognizes and binds the human soluble LDL receptor.
 3. Amonoclonal antibody according to claim 1, being the Mab expressed byhybridoma clone 12.6 deposited at the CNCM under No. 1-2390.
 4. Amonoclonal antibody according to claim 1, being the Mab expressed byhybridoma clone 28 deposited at the CNCM under No. I-1291.
 5. Amonoclonal antibody according to claim 1, being the Mab expressed byhybridoma clone 29.8 deposited at the CNCM under No. 1-2392.
 6. Amonoclonal antibody according to claim 1, being the Mab expressed byhybridoma clone 30 deposited at the CNCM under No. 1-2393.
 7. Amonoclonal antibody according to claim 1, being the Mab expressed byhybridoma clone 50.30 deposited at the CNCM under No. 1-2393. 8.Monoclonal antibodies according to any one of claim 3-7 the belong tothe immunoglobulin isotype IgG, or IgM.
 9. A method for the detectionand/or the quantitation of human LDLR which comprises the use of amonoclonal antibody according to any one of the preceding claims. 10.Monoclonal antibodies according to claim 1 capable of being used as apair in an ELISA.
 11. Monoclonal antibodies according to claim 10 beingmonoclonal antibodies generated by hybridoma clone 28 or clone 29.8. 12.Monoclonal antibodies according to claim 1 capable of identifying LDLRin Western Blot analysis.
 13. Monoclonal antibodies according to claim12 being monoclonal antibodies generated by hybridoma clone 12.6, clone28, clone 29.8, or clone
 30. 14. Monoclonal antibodies according toclaim 1 capable of neutralizing the antiviral biological activity ofr-hsLDLR.
 15. Monoclonal antibodies according to claim 14 beingmonoclonal antibodies generated by hybridoma clone 12.6 or clone 50.30.16. Monoclonal antibodies according to claim 1 capable of inhibiting thereplication of hepatitis c virus.
 17. Monoclonal antibodies according toclaim 16 being monoclonal antibodies generated by hybridoma clone 12.6,clone 28 or clone 29.8.
 18. The hybridoma clone 12.6 deposited at theCNCM under No.1-2390.
 19. The hybridoma clone 28 deposted at the CNCMunder No. 1-2391.
 20. The hybridoma clone 29.8 deposited at the CNCMunder No. 1-2392.
 21. The hybridoma clone 30 deposited at the CNCM underNo. 1-2393.
 22. The hybridoma clone 50.30 deposited at the CNCM underNo. 1-2393.
 23. A method for preparing a monoclonal antibody accordingto claim 1, comprising growing a cloned hybridoma comprising a spleencell from a mammal immunized with hsLDL and a homogenic or heterogeniclymphoid cell in liquid medium or mammalian abdomen to allow thehybridoma to produce and accumulate the monoclonal antibody.
 24. Amethod according to claim 21 wherein the LDLR immunogen is highlypurified human LDLR.
 25. A method for purifying human LDLR whichcomprises contacting a material containing human LDLR with a monoclonalantibody according to any one of claims 1-8.